Structural Basis for Aza-Glycine Stabilization of Collagen

Abstract

Previously, we have demonstrated that replacement of the strictly conserved glycine in collagen with aza-glycine provides a general solution for stabilizing triple helical collagen peptides (Chenoweth, D. M.; et al. J. Am. Chem. Soc. 2016, 138, 9751 ; 2015, 137, 12422 ). The additional hydrogen bond and conformational constraints provided by aza-glycine increases the thermal stability and rate of folding in collagen peptides composed of Pro-Hyp-Gly triplet repeats, allowing for truncation to the smallest self-assembling peptide systems observed to date. Here we show that aza-glycine substitution enhances the stability of an arginine-containing collagen peptide and provide a structural basis for this stabilization with an atomic resolution crystal structure. These results demonstrate that a single nitrogen atom substitution for a glycine alpha-carbon increases the peptide's triple helix melting temperature by 8.6 °C. Furthermore, we provide the first structural basis for stabilization of triple helical collagen peptides containing aza-glycine and we demonstrate that minimal alteration to the peptide backbone conformation occurs with aza-glycine incorporation.

Rules for the design of aza-glycine stabilized triple-helical collagen peptides

“…Averaging 4 angles of Gly residues from collagen structures in the PDB gives a value of À69 AE 5 while averaging j angles gives a value of À176 AE 5 or 173 AE 5 . 32 In our structure, the Gly residues adjacent to the Xaa NmetG residue adopt conformations closely matching the PDB average Gly (4, j) angles with average values of (À68 , 176 ) (Fig. 5d and Table S4 †).…”

“…1a). [30][31][32][33] In most cases, incorporation of this aza-amino acid within a simple model system results in increased triple-helical thermal stability as assessed by thermal melting studies. The azGly residues stabilize the collagen triple helix in two primary methods: the formation of additional interstrand hydrogen bonds via the azGly residue's NHa and the restricted conformational exibility compared to Gly residues.…”

Deciphering the Backbone Noncovalent Interactions that Stabilize Polyproline II Conformation and Reduce cis Proline Abundance in Polyproline Tracts

“…We then investigated the φ and ψ angles of these hits to see if these hits fall within the PPII helical range according to our prediction. Interestingly, the φ and ψ angles of these molecules primarily fell in the PPII (−75, 150°) and the mirror image PPII regions (poly-Gly II) (75, −150°), with a few molecules having φ and ψ in the aza-Gly region 77 (Figure 4C and Table S17). NBO analyses of selected molecules obtained from the CSD search confirmed the ddda n → π* nature of their CO•••CO interactions (Table S18− A few oxygen atoms that participated in the single CO•••CO interaction instead of two were the ones that were involved in interchain HB with the neighboring strands to form the triple helix (Figure 5E).…”

Incorporation of Aza-Glycine into Collagen Peptides

“…Compared to their natural analogues, aza-amino acids exhibit substantially different properties including restricted conformational flexibility and an absence of chirality at the α-position. , Several strategies have been developed for aza-amino acid incorporation and their derivatization. ,− One of the most common approaches for incorporation of aza-amino acid residues utilizes phosgene or phosgene-like carbonyl equivalents to either activate a substituted hydrazine or the terminal amino group of the growing peptide chain on resin (Figure b). In our hands, the synthesis of aza-glycine (azGly, azG)-containing peptides can be difficult and low yielding due to reduced reactivity of the activated hydrazine compound compared to that of an activated amino acid. , This synthetic approach can be quite slow, with many groups reporting long reaction times ranging from 6 to 24 h for similar strategies. ,,,− …”